Construction And Expression Of Divalent H-2D~b/IgG2b Molecule

Multiple sclerosis (MS) is the commonest autoimmune disorder of the central nervous system (CNS). Studies of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, focus on the contribution of CD4⁺ myelin-specific T cells. However, CD8⁺ T cells specific for myelin antigens have been isolated from peripheral blood of MS patients, and infiltrating CD8⁺ T cells are present at higher frequency than CD4⁺ T cells in the CNS, particularly in active demyelinating lesions. Therefore, to invastigate the pathophysiologic mechanism of MS and EAE, studies have become fcous on the role of CD8⁺ T cells. Blocking autoreactive CD8⁺ T cells contributes to prevention the occuring and developing of EAE. Previous studies have shown that soluble peptide/major histocompitibility complex class (pMHC) dimer can be used to specific modulate the immune response by interacted with T cell receptors(TCR). In this study, we construct and express of divalent H-2D^b/IgG2b molecule on the base of the protophase work. Soluble divalent MOG35-55/H-2D^b molecule, which is prepared by the peptide MOG35-55 loading to divalent H-2D^b/IgG2b molecule, may help to further develop of inducing T cells autoreactive tolerance and provide evidence for blocking the process of EAE. This work contains the following two major parts:1. Construction of divalent H-2D^b/IgG2b moleculeThe gene of H-2D^bαchain was amplified by RT-PCR method from total RNA of leukomonocyte which came from C57BL/6(B6) mouse(H-2b). H-2D^bαchain was joined with Fc fragments of mouse IgG2b through the restriction site and formed H-2D^b/IgG2b recombinant gene. Then, the recombinant fragment and mouse light chainβ2m were inserted into insect baculovirus expression vector pFastBac^TMDual downstream of two promoters Pp10 and PpH. The recombinant plasmid named [H-2D^b/IgG2b]+pFastBac^TMDual+β2m was confirmed by PCR and enzyme digestion. The entire fusion gene was sequenced to ensure that no spurious mutations were introduced during the PCR.2. Expression, purification and identification of divalent H-2D^b/IgG2b moleculeThe recombinant plasmid constructed was transformed to Escherichia coli DH10Bac to obtain the recombinant shuttle plamid Bacmid+[H-2D^b/IgG2b]. The shuttle plamid was transfected into the insect cell line Sf9 by cellfection and purified by SPA affinity chromatography. The purified H-2D^b/IgG2b dimer were detected by Sandwich ELISA and Flow cytometry (FCM) with conformationally-dependent H-2D^b specific antibody and anti-IgG2b Fc antibody, which suggests the purified H-2D^b/IgG2b dimer with intact comformation. Western blotting by specific antibody shows the H-2D^b/IgG2b fusion protein with correct molecule weight about 61KDa.